A requirement for proper cellular function is RNA homeostasis, a process maintained through a balance between transcription and RNA turnover. The eukaryotic exosome is the cell's major 3'5'exoribonuclease, with multi-faceted roles that include RNA degradation, processing, and quality control in the cytoplasm and nucleus. The non-catalytic core of the Saccharomyces Cerevisiae exosome is composed of 9 distinct and essential subunits that likely form a ring-shaped structure (Exo9). This core is associated with a processive, hydrolytic exoribonuclease, Rrp44 in the nucleus and cytoplasm, and with the distributive, hydrolytic exoribonuclease, Rrp6 in the nucleus. Rrp6 activities are known to be important for processing structured substrates such as ribosomal RNA. The precise role of Exo9 in modulating the activities of its catalytic subunits, Rrp44 and Rrp6 is unknown. An outstanding question in the field is whether all RNA substrates targeted by the exosome are threaded through the pore, as they are in RNA degradation assemblies from archaea and bacteria. Because the architecture of these complexes is structurally conserved, it is tempting to speculate that the eukaryotic exosome operates through a similar mechanism. Additionally, biochemical evidence shows that Rrp6 activity is predominant relative to Rrp44 in Exo11 in vitro. Therefore, another question pertains to the accessibility of these two catalytic subunits on Exo9, and their positions relative to each other. To address these questions, X-ray crystallography will be used for structure determination of the 9-subunit core in complex with its catalytic subunits and a bound RNA substrate, providing mechanistic insight to how architecture contributes to function. Furthermore, RNA decay assays will kinetically describe how the core modulates the activities of Rrp44 and Rrp6, and how, if at all, the pore is involved in RNA decay. Recombinant yeast exosome proteins that lack affinity tags will be purified and used for biochemical reconstitution. This strategy will facilitate reconstitution of the 9-, 10-, and 11-subunit exosome complexes that better mimic their cellular counterparts in addition to providing samples more amenable to crystallization through surface entropy reduction. PUBLIC HEALTH RELEVANCE: The balance between RNA transcription and decay is essential for proper cellular function. The RNA exosome is involved in RNA processing, maturation, degradation, and quality control. Disruption of exosome-mediated processes are associated with diseases such as cancer and autoimmune disorders. This project will address the mechanism by which the RNA exosome regulates these key activities.